The purpose of this project is to understand at the molecular level the intercellular interactions that control the conjugation response of the yeast Saccharomyces cerevisiae, a relatively simple developmental and morphogenetic sequence. The research will focus primarily on the expression and mode of action of alpha-factor, the mating-specific oligopeptide pheromone released by alpha haploid cells. These studies will include: (a) Characterization of the gene products involved in modulation of adenylate cyclase activity by alpha-factor. Radioactive derivatives of alpha-factor and matrix bound alpha-factor will be used to isolate a pheromone "receptor". Cloned DNA will be used to identify the STE5 gene product, which is required for pheromone inhibition of the enzyme. Other elements of the adenylate cyclase complex, including catalytic subunit and additional regulatory components like calmodulin, will be purified and used to reconstitute the system in vitro. (b) Investigation of the role of protein phosphorylation in the control of cell division by alpha-factor. In vivo targets for covalent modification among microtubule components and other associated nuclear proteins, as well as in plasma membrane, will be analyzed by two-dimensional polyacrylamide gel electrophoresis. The activities and distribution of cyclic AMP-dependent and independent protein kinases will be measured in vitro. (c) Examination of the mechanism of alpha-factor biosynthesis. Immunological and biochemical methods will be used to identify precursor forms of the pheromone. Direct transformation of certain pheromone-deficient mutants with cloned yeast DNA, immunological screening, and preparation of a complementary oligonucleotide probe will be used in attempts to isolate the alpha-factor structural gene. (d) Selection of a large collection of alpha-factor-deficient alpha cell mutants and alpha-factor-insensitive a cell mutants. Immunological and biochemical examination of such mutants will aid analysis of the cellular components involved in the synthesis and function of alpha-factor.